Artemisia annua L.  has received increasing attention due to the fact that the plant produces the sesquiterpenoid endoperoxide artemisinin, which today is widely used for treatment of malaria. The plant produces relatively small amounts of artemisinin and a worldwide shortage of the drug has led to intense research in order to increase the yield of artemisinin. In order to improve our understanding of terpene metabolism in the plant and to evaluate the competition for precursors, which may influence the yield of artemisinin, we have used qPCR to estimate the expression of 14 genes of terpene metabolism in different tissues.Recently, epi-cedrol synthase was highly expressed in old leaves. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase exhibited lower expression in old leaves compared to other tissues. Farnesyldiphosphate synthase, squalene synthase, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase showed only modest variation in expression in the different tissues, while expression of 1-deoxy-D-xylulose-5-phosphate synthase was 7-8-fold higher in flower buds and young leaves compared to old leaves.The four genes of the artemisinin biosynthetic pathway  reductase and aldehyde dehydrogenase 1) showed remarkably higher expression (between ~40- to ~500-fold) in flower buds and young leaves compared to other tissues . Further, dihydroartemisinic aldehyde reductase showed a very high expression only in hairy root cultures. Germacrene A and caryophyllene synthase were mostly expressed in young leaves and flower buds while Four genes of artemisinin biosynthesis were highly expressed in flower buds and young leaves (tissues showing a high density of glandular trichomes). The expression of dihydroartemisinic aldehyde reductase has been suggested to have a negative effect on artemisinin production through reduction of dihydroartemisinic aldehyde to dihydroartemisinic alcohol. However, our results show that this enzyme is expressed only at low levels in tissues producing artemisinin and consequently its effect on artemisinin production may be limited. Finally, squalene synthase but not other sesquiterpene synthases appears to be a significant competitor for farnesyl diphosphate in artemisinin-producing tissues. Artemisia, belonging to the Asteraceae family, contains a large number of aromatic plants. During the latest decades, A. annua L.  has received increasing attention due to the fact that the plant produces the sesquiterpenoid endoperoxide artemisinin, which today is widely used for the treatment of malaria [The genus  malaria . The pla malaria . Artemis malaria . The api malaria . This haA. annua are mono- and sesquiterpenes produced in trichomes [A. annua. In fact, so far five different sesquiterpene synthases (epi-cedrol synthase (ECS) [A. annua since other classes of sesquiterpenes such as eudesmanes (e.g. β-selinene), guaianes (e.g. α-guaiene), longipinanes (e.g. β-longipinene) and santalanes  have been isolated from the plant [The major components of the essential oil from richomes . The larse (ECS) , amorphase (ECS) ; β-caryose (ECS) , germacrse (ECS)  and β-fase (ECS)  have beehe plant . All theA. annua. The first committed step in artemisinin biosynthesis is the conversion of FDP to amorpha-4,11-diene by ADS [Figure e by ADS ,11. In te by ADS . This ene by ADS . It has e by ADS  and dihye by ADS  have shoe by ADS  and the e by ADS . It has e by ADS ,17.The conversion of dihydroartemisinic acid to artemisinin is believed to be a non-enzymatic spontaneous reaction . In a siA. annua [Recently, a dihydroartemisinic aldehyde reductase (RED1) has been cloned from A. annua . This enA. annua appears to be high. At least two chemotypes with different compositions of the essential oil during the vegetative period have been described [in planta of artemisinic acid to dihydroartemisinic acid or vice versa [in planta [The genetic variation within escribed . One chece versa . It has n planta .A. annua and to evaluate the competition for precursors, which may influence the yield of artemisinin in the plant, we have used qPCR to estimate the expression of genes of terpene metabolism in different plant tissues. cDNA made from flower buds, young leaves, old leaves, stems, roots and hairy roots has been used as templates for the qPCR studies. In total, 14 different transcripts have been monitored using three reference genes. The different tissues have also been studied by fluorescence microscopy to determine trichome densities.In order to increase our understanding of terpene metabolism in the plant A. annua have been extensively studied and it is well established that the production of specific terpenoids, including artemisinin, takes place within the secretory cells of such trichomes [A. annua were visualized by fluorescence microscopy .The glandular trichomes of richomes ,22. Furtrichomes . In orde2 while the corresponding number for old leaves was 130 ± 25 trichomes/cm2. The original number of trichomes seem to be spread out on a larger surface as the leaf expands with age.From Figure In order to examine the gene expression of 14 enzymes of terpene metabolism and three reference transcripts by qPCR, 18 primer pairs were synthesized as listed in Table A. annua and the results are summarized in Table The gene expression of the 14 enzymes of terpene metabolism was studied in different tissues of CT-values for this transcript in these tissues were relatively low (23.8-24.7) indicating that HMGR was expressed at relatively high levels  have been cloned from A. annua. The two FDS genes from A. annua show 96.6% nucleotide sequence identity and the segment that was amplified with the primers used  plays a central role in the conversion of IDP and DMADP, produced by the mevalonate pathway, to various terpenoids such as sterols and sesquiterpenes. A small gene family encodes isoenzymes of FDS in plants. l stages . It is ioduction ,30 indicelopment  and is ant [FDS1 ,32 and FE)-butenyl 4-diphosphate reductase (HDR), have been studied by qPCR in this paper. Our results show that the expression of DXS was 7-8-fold higher in flower buds and young leaves compared to old leaves , which in Arabidopsis is involved in jasmonic acid biosynthesis [A. annua (Genbank accession number EU848577) shows a very high similarity to DBR2, and in our initial attempt to determine the relative expression of DBR2, a primer pair  due to its low abundance in flower buds. Using published kcat-values for the different enzymes, we may calculate their relative turnover potential in planta as summarized in Table cat-value is available and therefore this enzyme is not included in this estimation. It may be concluded that ADS is a rate-limiting enzyme due to its low kcat-value and that the other trichome-specific enzymes DBR2 and ALDH1 . Finally, since dihydroartemisinic acid accumulates in this chemotype, we may conclude that the activity of DBR2 is significantly higher than that of CYP71AV1. The fact that DBR2 is a soluble cytosolic protein while CYP71AV1 is embedded in the ER may influence the metabolic flow and the rate of intracellular transport of intermediates has to be considered. At present, we do not have any information on the transport of intermediates of artemisinin biosynthesis within cells. It would be interesting to study the ratio of DBR2 and CYP71AV1 in the two different chemotypes of A. annua since these enzymes are the key enzymes for the formation of dihydroartemisinic acid and artemisinic acid, respectively.From Figure oduction  indicatiet al. [m for dihydroartemisinic aldehyde, low relative turnover potential and only partial localization to trichomes. Expression of RED1 in flower buds, young leaves and stems was relatively low and just a fraction of that observed in old leaves , results in about 45- and 25-fold higher turnover potential, of FDP to products, for SQS, as compared to the turnover potential of ADS in flower buds and young leaves, respectively. The fraction of SQS expressed in trichome cells may channel part of available FDP into sterols and thereby lower the yield of artemisinin. Zhang et al. showed that down-regulation of SQS by hairpin-RNA-mediated gene silencing in A. annua resulted in a 3-fold increased artemisinin production [A key enzyme in the biosynthesis of sterols and triterpenes is squalene synthase (SQS), which condensates two molecules of FDP to squalene. The expression of SQS varied only modest between different tissues in our qPCR study. SQS is a microsomal enzyme and difficult to purify and characterize. Consequently, there is no reported kt enzyme . The A.  tabacum . As seenoduction .A. annua. The qPCR data presented in this paper demonstrate that four genes of the artemisinin biosynthetic pathway  showed remarkably higher expression (between 40- to 500-fold) in flower buds and young leaves compared to other tissues   Figure . These hi.e. flower buds and young leaves). Consequently, their influence on artemisinin yield appear to be relatively limited and downregulation of other sesquiterpene synthase(s) will most likely not affect artemisinin production in A. annua.Our aim was also to evaluate the competition for precursors, which may influence the yield of artemisinin in the plant. The expression of other sesquiterpene synthases was much lower than ADS in tissues producing artemisinin precursors . This variety is a high dihydroartemisinic acid chemotype (the ratio artemisinic acid to dihydroartemisinic acid is 0.36 [	Seeds of  is 0.36 ). PlantsArtemisia annua L. hairy root cultures, kindly provided by Dr. Kanyaratt Supaibulwatana, Mahidol University, Bangkok, were grown in MSMO medium  pH 5.8, supplemented with 3% (w/v) sucrose at room temperature on a rotary shaker at 75 rpm.Flower buds, young leaves, old leaves, stems, roots and hairy roots were collected separately from 5-6 months old plants after induction of flower buds, frozen in liquid nitrogen, ground to a fine powder in a mortar and used for RNA extraction.RNA extraction was performed using Purelink™ Plant RNA Reagent kit  for small scale RNA isolation according to the manufacturer's instructions. Frozen plant tissue powder (100 mg) was used for each RNA extraction. The RNA was DNase treated using DNase I  to remove remaining genomic DNA.18 primer. The RNA was removed from the first strand cDNA by RNase treatment using RNase H (Fermentas) according to the manufacturer's instructions.RNA (1 μg) was reverse transcribed using RevertAid™ H Minus-MuLV reverse transcriptase (Fermentas) primed with 0.5 μg oligo(dT)®Green PCR Master Mix (Applied Biosystems) and 2 pmol of each primer. The qPCR cycling was performed at 50 °C (2 min), 95 °C (10 min), 40 cycles at 95 °C (15 s), 60 °C (1 min) and finally a dissociation stage at 95 °C (15 s), 60 °C (1 min), 95 °C (15 s). The dissociation stage was performed to determine the PCR product size and to detect possible primer dimers. Triplets of all samples were run, and a negative control of the Master Mix in addition of primers was performed in all qPCR runs.The qPCR was performed using specific primers Table  on a 750CT) values and efficiency values given by the Linreg software were used for further analysis. The BestKeeper software [Any outliers in the triplets were detected by Grubbs test and removed. The mean efficiency of the amplicons was calculated by the program Linreg v. 12.1 based on the log linear phase of the amplification curve . Baselinsoftware  was usedsoftware .A. annua plant and microscopy studies were performed to compare the amount of trichomes on different plant tissue. All micrographs were captured using a Nikon e400 C-SHG1 fluorescence microscope equipped with digital camera, using light microscopy and filter settings for FITC  and Texas Red . Autofluorescence was separated in the red and green channels and brightness adjusted using NIS-elements imaging software v. 2.20 . All tissue images have been captured using the same magnification .Flower buds, young leaves, old leaves, stems and roots were selected from the same epi-cedrol synthase; FDP: farnesyldiphosphate; FDS: farnesyl diphosphate synthase; GAS: germacrene A synthase; GDP: geranyldiphosphate; HDR: hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; HMGR: 3-hydroxy-3-methyl-glutaryl-CoA reductase; IDP: isopentenyldiphosphate; MSMO: Murashige and Skoog medium with minimal organics; OPR3: 12-oxophytodienoate reductase; PAL: phenylalanine ammonia lyase; RED1: dihydroartemisinic aldehyde reductase; SQS: squalene synthase.AA: artemisinic aldehyde; AAOH: artemisinic alcohol; AD: amorpha-4,11-diene; ADS: amorpha-4,11-diene synthase; ALDH1: aldehyde dehydrogenase 1; CPR: cytochrome P450 reductase; CPS: β-caryophyllene synthase; CYP71AV1: amorphadiene-12-hydroxylase; DBR2: artemisinic aldehyde Δ11(13) reductase; DHAA: dihydroartemisinic aldehyde; DXR: 1-deoxy-D-xylulose 5-phosphate reductase; DXS: 1-deoxy-D-xylulose 5-phosphate synthase; ECS: LO planned the experimental setup and PB was involved in the study design. AL prepared the plants. LO and AE carried out the RNA extraction, cDNA synthesis and qPCR analysis and analyzed the data. LO and AL performed the fluorescence microscopy. PB, LO, AL drafted and wrote the manuscript. All authors have read and approved the final manuscript.


Wistar rats were subjected to the defensive burying test, elevated plus maze, and open field test. In different groups, three GABAAreceptor antagonists were administered 30 min before FAT-M administration, including the competitive GABA binding antagonist bicuculline (1 mg/kg), GABAA benzodiazepine antagonist flumazenil (5 mg/kg), and noncompetitive GABAA chloride channel antagonist picrotoxin (1 mg/kg). The FAT-M exerted anxiolytic-like effects in the defensive burying test and elevated plus maze, without affecting locomotor activity in the open field test. The GABAA antagonists alone did not produce significant changes in the behavioral tests. Picrotoxin but not bicuculline or flumazenil blocked the anxiolytic-like effect of the FAT-M. Based on the specific blocking action of picrotoxin on the effects of the FAT-M, we conclude that the FAT-M exerted its anxiolytic-like effects through GABAA receptor chloride channels.Human amniotic fluid and a mixture of eight fatty acids (FAT-M) identified in this maternal fluid (C12:0, lauric acid, 0.9  The actnt drugs . Consequoperties , 4.A receptor antagonists, such as bicuculline, flumazenil, and picrotoxin, are currently used to identify the precise sites of action of drugs on the GABAA receptor. These antagonists are able to block the anxiolytic-like effects of diazepam and some neurosteroids [A receptor chloride channels and blocks the anxiolytic-like effects of diazepam, 4′-chlorodiazepam, progesterone, and allopregnanolone [A receptors [Several GABAsteroids , 5, 6. Tsteroids , among osteroids , 8, 9. Pnanolone , 10, 11,eceptors .cis, oleic acid; C18:1trans, elaidic acid; C18:2, linoleic acid) that were consistently detected in human amniotic fluid, colostrum, and milk. In addition to its anxiolytic effects [A receptors, given that some FATs modulate the opening of GABAA receptor chloride channels in vitro [Recently it was demonstrated that human amniotic fluid and a mixture of eight fatty acids (FAT-M) contained therein produce anxiolytic-like effects similar to diazepam in male and female adult Wistar rats . The FAT effects , the FAT effects . The anxin vitro , but thiA site of action involved in the anxiolytic-like effects of a FAT-M using bicuculline (a competitive antagonist of the GABAA receptor), flumazenil (a blocker of the benzodiazepine recognition site), and picrotoxin (a noncompetitive antagonist of chloride channels).Therefore, the present study confirmed the anxiolytic-like effect of a FAT-M using two validated experimental animal models currently used to test the effectiveness of clinically effective anxiolytics, the defensive burying test  and elevGuide for the Care and Use of Laboratory Animals . The prMale Wistar rats were obtained from a local strain supplied by Harlan . They were housed in local housing facilities at a mean temperature of 25 ± 2°C with a 12 h/12 h light/dark cycle (lights on at 7:00 AM). All of the rats included in the study were approximately 2 months old, weighed 250–300 g, and five to six rats were housed per cage in acrylic boxes (44 cm width × 33 cm length × 20 cm height) with ad libitum access to food  and purified water. All of the experiments were performed during the light period (approximately 12:00 PM).An acrylic box (27 cm width × 17.5 cm length × 30 cm height) with the floor covered by a 5 cm bed of fine sawdust  was placed inside a noise-isolated box . An electrode  protruded 2 cm above the sawdust bed horizontally from one wall of the box (17.5 × 30 cm) , 19. TheThe apparatus was constructed of wood and situated in a brightly lit room (40 lux). The apparatus consisted of two opposite open and closed arms set in a perpendicular configuration. The open and closed arms were painted white and black, respectively. The dimensions of the open arms were 50 cm length × 10 cm width and the closed arms were 50 cm length × 10 cm width × 40 cm height. The entire maze was elevated 50 cm from the floor. The rats were placed in the center of the maze, facing an open arm, and the time spent on and number of entries into the open arms was recorded . The totversus peripheral exploration.To evaluate the effects of the treatments on spontaneous locomotor activity, which could interfere with performance in the defensive burying test and elevated plus maze, the rats were subjected to a 5 min test in the open field after the defensive burying and elevated plus maze tests. We used an automated motor activity monitor  in a Perspex box . A total of 32 infrared beams, 16 each on perpendicular walls, were mounted 3 cm above the box frame floor and connected to an interface , and the data were sent to a computer. For data analysis, the floor of the cage was virtually divided into five zones . The total number of entries into the zones , time spent active, and time spent resting were recorded. Because of the relatively small cage, we did not compare central After each experimental session, the open field was carefully cleaned and deodorized with a 5% ethanol cleaning solution. Five minutes elapsed between each test to allow the scent of the substances to dissipate. The doses and pretreatment schedules were based on previous reports , 7, 21 tA antagonists  and another for the FAT-M (vehicle-2: 96% propylene glycol and 4% ethanol). All of the chemical compounds were purchased from Sigma-Aldrich .We used two vehicles, one for the GABAThe FAT-M preparation and treatment schedule were based on previous reports . The FATA antagonists on anxiety-like behavior and open field activity, four independent groups were evaluated in the defensive burying test and subsequently open field test. The treatment conditions included four combinations: vehicle-1 before vehicle-2 , bicuculline before vehicle-2 , flumazenil before vehicle-2 , and picrotoxin before vehicle-2 . Other four independent groups (n = 8 rats per group) received a similar treatment schedule but were tested in the elevated plus maze and subsequently open field test.To identify the intrinsic activity of the GABAn = 11), vehicle-1 before FAT-M , bicuculline before FAT-M , flumazenil before FAT-M , and picrotoxin before FAT-M . The elevated plus maze test included another five independent groups (n = 8 rats per group) that received similar treatments. The open field test was conducted less than 5 min after the elevated plus maze test.The defensive burying test included five independent groups that received five different combinations: vehicle-1 before vehicle-2 . Values of A antagonists alone did not affect burying latency  or cumulative burying time  in the defensive burying test. The antagonists also did not affect the number of crossings , activity time , or resting time  in the open field test. Likewise, the antagonists did not produce significant changes in the elevated plus maze, including the time spent in the open arms , number of entries into the open arms , total number of entries into the arms , and percentage of entries into the open arms . Similarly, no significant changes were found in the open field test, including the number of crossings , activity time , and resting time . The GABAF4,56 = 3.45, P = 0.014). The post hoc test showed that burying latency was significantly longer in the FAT-M groups compared with the vehicle-1 + vehicle-2 group, but no significant differences were detected in the FAT-M groups pretreated with GABAA antagonists compared with the vehicle-1 + vehicle-2 group . The post hoc test revealed that cumulative burying time was significantly shorter in the FAT-M, bicuculline + FAT-M, and flumazenil + FAT-M groups than in the vehicle-1 + vehicle-2 group. However, the picrotoxin + FAT-M group was not significantly different from the vehicle-1 + vehicle-2 group . The post hoc test revealed that the time spent in the open arms was longer in the FAT-M, bicuculline + FAT-M, and flumazenil + FAT-M groups than in the vehicle-1 + vehicle-2 and picrotoxin + FAT-M groups, which displayed similar values . The post hoc test revealed that this variable was higher in the FAT-M, bicuculline + FAT-M, and flumazenil + FAT-M groups than in the vehicle-1 + vehicle-2 and picrotoxin + FAT-M groups . The percentage of entries into the open arms was greater in the FAT-M, bicuculline + FAT-M, and flumazenil + FAT-M groups than in the vehicle-1 + vehicle-2 and picrotoxin + FAT-M groups . Similarly, no significant differences were detected in activity time  or resting time  is inversely related to the rat's reactivity. The time spent burying  is an indicator of anxiety as discussed by Treit . “AnxiouA receptor antagonists tested in the present study.The elevated plus maze is widely used to explore anxiety-like behavior and the anxiogenic- or anxiolytic-like effects of drugs . AnxietyA receptor chloride ion channels, but not benzodiazepine or GABA binding sites, participate in the anxiolytic-like effects of the FAT-M. Certainly, some FATs  seemingly participate in the regulation of chloride ion channels [A receptors [The present results suggest that GABAchannels . Intereseceptors , thus moeceptors  by accumeceptors .A chloride ion channels [Chloride channels participate in the actions of substances with anxiolytic potency –28. Othechannels –32. At pchannels , 34. Majchannels , suggestA antagonists alone or the FAT-M at the doses tested produced any significant changes on motor activity, similar to previous reports [A antagonists do not appear to be associated with nonspecific effects of the treatments on spontaneous locomotion.Finally, some drugs are able to produce nonspecific changes in spontaneous locomotor activity that may interfere with performance in the defensive burying test and elevated plus maze. The open field test was conducted after the aforementioned behavioral tests, allowing us to exclude possible nonspecific locomotor effects of antagonists or FAT treatments. In the open field test, we found that none of the GABA reports , 13, 35.A possible limitation of the present study was that the FAT-M was administered in addition to the rats' normal diet , possibly providing an additional source of energy that may impact spontaneous behavior. However, we did not observe any change in locomotion associated with the FAT-M treatment, similar to previous reports , 36. AltA receptors, specifically chloride channels, providing additional evidence of the anxiolytic-like effects of FATs and the possible site of action on GABAA receptor of the FAT-M used in the present study. However, the participation of other receptors linked to chloride ion channel  could not to be discarded, which needs to be explored in particular studies.In conclusion, the anxiolytic-like effects of the FAT-M involved actions on GABA


G. hollisae thermostable direct hemolysin (Gh-TDH) is produced by most strains of G. hollisae. This toxin has been reported to be absorbed in the intestines in humans. Secondary liver injury might be caused by venous return of the toxin through the portal system. We aimed to firstly analyze the in vitro and in vivo hepatotoxicity of Gh-TDH.Liver cells (primary human non-cancer cell and FL83B mouse cells) were treated and mice (BALB/c) were fed with this toxin to investigate its hepatotoxicity. Morphological examination and cytotoxicity assays using liver cells were also performed. Fluorescein isothiocyanate-conjugated toxin was used to analyze the localization of this protein in liver cells. Mice were subjected to liver function measurements and liver biopsies following toxin treatment and wild-type bacterial infection. PET (positron emission tomography)/CT (computed tomography) images were taken to assess liver metabolism during acute injury and recovery.Grimontia hollisae showed elevated levels of transaminases and damage to the periportal area, respectively. The PET/CT images revealed that the reconstruction of the liver continued for at least one week after exposure to a single dose of the toxin or bacterial infection.The effect of hepatotoxicity was dose and time dependent. Cellular localization showed that the toxin was initially located around the cellular margins and subsequently entered the nucleus. Liver function measurements and liver biopsies of the mice following treatment with toxin or infection with wild-type The hepatotoxicity of Gh-TDH was firstly demonstrated. The damage was located in the periportal area of the liver, and the liver became functionally insufficient. Vibrio species have been observed in large populations throughout the world, particularly in Asia, the United States, and Africa V. cholera and V. parahaemolyticus are the major etiological agents of vibriosis, which is associated with the ingestion of raw, undercooked, or contaminated seafood Grimontia hollisae (previously named V. hollisae) has been frequently reported to cause diseases in humans, including severe gastroenteritis, hypovolemia, and septicemia following the consumption of shellfish or oysters G. hollisae has not been thoroughly characterized. Although several specific laboratory examinations have detected G. hollisae, its incidence is likely highly underestimated because detection techniques are infrequently used throughout the world Diseases caused by different Vibrio species, including V. cholera non-O1, V. parahaemolyticus, V. mimicus, V. alginolyticus, and G. hollisaetdh gene sequences and pathogenicities tdh gene is present in all strains of G. hollisae but not in all Vibrio species G. hollisae TDH (Gh-TDH) are not well known. Some studies have reported that Gh-TDH is detoxified by aggregation into fibrils after heating at 60–70°C; the protein can be reversibly refolded into the toxic native form by rapid cooling after unfolding at higher temperatures in vivo and in vitro to provide insights into the acute injury and recovery stages of THD-induced hepatotoxicity in living animals.The toxin thermostable direct hemolysin (TDH) is composed of 165 amino acid residues and exhibits biological activities that include hemolytic activity, cytotoxicity, and enterotoxicity G. hollisae strain ATCC 33564 was obtained from the Culture Collection and Research Center . Phenyl Sepharose 6 Fast Flow and protein molecular weight standards were purchased from GE Healthcare . The protein assay kit was obtained from Bio-Rad . Protein purification chemicals were obtained from Calbiochem .Molecular cloning, protein expression, and purification of Gh-rTDH were performed according to previous publications 50 determination . Cellular morphology in the experimental group was observed microscopically at 4 time points . Cells treated with PBS (mixed with culture medium) were used as the control group and were observed at the same time points as the experimental group. The cytoviability of human liver cells and FL83B cells was measured by MTT assay at 4 treatment durations . In the MTT assay, cells were treated with PBS as a control and with Gh-rTDH at different concentrations . All experiments were independently performed five times.FL83B (BCRC 60325) and primary human non-cancer cells  were cultured for use in these studies. Following surface attachment, the cells were treated with Gh-rTDH at a concentration of 1 µg/ml for 24 hr at 37°C; the treatment dose was determined by using the initial results from the IC4 cells/well) were independently treated with 10 µg/ml Gh-rTDH-FITC for 20 and 40 min, washed 3 times with PBS, and stained with propidium iodide (PI) for 5 min in the dark. Images were acquired by confocal microscopy (wavelength: λex = 488 and λem = 650 nm).To investigate the localization of Gh-rTDH after its entry into FL83B cells, Gh-rTDH was conjugated with fluorescein isothiocyanate (FITC) to produce Gh-rTDH-FITC, and reactions were performed using the FluoReporter FITC Protein Labeling Kit (Molecular Probes) according to the manufacturer’s protocol. Two batches of cells . All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.A total of 114 six-week-old female mice were obtained from the National Laboratory Animal Center of Taiwan for the analysis of 50 determination . All mice were treated with the same volume (200 µl) and the same treatment time (10∶00 a.m.) via gastric tubes without volume loss . A total of 100 µl of whole blood was withdrawn from the orbital vascular plexus of each mouse through a capillary tube with no analgesics. Samples were taken at 8 time points: before treatment with PBS or Gh-rTDH and 4, 8, 16, 32, 64, 128, and 256 hr after treatment with PBS or Gh-rTDH. The blood samples were analyzed for the continuation of liver function as assessed by glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), total/direct/indirect bilirubin, albumin, and globulin (Reagents Beckman Coulter). One-way ANOVA analysis was used to analyze the significance of differences between each treatment/time point. All analyses were performed with the SPSS statistical package for Windows .A total of 25 mice were assigned to one of 5 groups (n = 5 for each group). One group served as a control group and was administered PBS; the other 4 groups were administered different doses of Gh-rTDH  in a single treatment. The dosage that might initiate organ injury in animals has not been reported . Therefore, the treatment dosages were carefully determined and modified according to the initial results of the ICA total of 20 mice were assigned to one of 4 groups (n = 5 in each group). One group served as the control group and was treated with PBS. The other 3 groups were treated with Gh-rTDH at doses of 1, 10, and 100 µg in a single administration via a gastric tube. A total of 100 µl of whole blood was withdrawn from each mouse at 5 time points: before treatment with PBS or Gh-rTDH and 4, 16, 64, and 256 hr after treatment with PBS or Gh-rTDH. Nephrotoxicity was assessed by determining the creatinine levels in the blood samples , and cardiotoxicity was assessed by analyzing the levels of CK-MB  and troponin I (ADVIA Centaur TnI-Ultra Ready Pack).A total of 9 mice were assigned to one of 3 groups which were treated with PBS, 10 µg Gh-rTDH, or 100 µg Gh-rTDH (n = 3 in each group) in a single administration via a gastric tube. The livers of all mice were biopsied after 8 hr of treatment. Samples were prepared from tissue that was harvested at the time of sacrifice and subjected to H&E staining.18F-FDG PET/CT scan was used to detect liver cell glucose metabolism in living animals after exposure to Gh-rTDH to monitor trends in glucose metabolism . 18F-FDG is an analog of glucose that can be used to measure glucose metabolism in organs and cells. A total of 60 mice were assigned to one of 4 dosage groups, and each group (n = 15) was treated with PBS or 1, 10, or 100 µg of Gh-rTDH in a single administration. Within each dosage group, mice were further sub-grouped to receive 18F-FDG PET/CT scans over time; examinations were performed at 8, 72, and 168 hr (n = 5 for each time group) after treatment with Gh-rTDH. For this study, 0.07 mCi of 18F-FDG was administered to each mouse by tail vein injection. Imaging was performed under appropriate general anesthesia (Isoflurane) one hour after 18F-FDG injection. In this study, each mouse did not receive a 18F-FDG PET/CT scan at each time point. The recurring general anesthesia might cause hepatotoxicity, which could influence the results of the study. For this analysis, the 18F-FDG uptake value was calculated by using a region of interest approach (ROI). The ROIs of liver and muscle (left foot) were determined by a semi-quantitative method using the ratio of liver/muscle 18F-FDG uptake.A G. hollisae (wild type), E. coli-TOPO-tdh, and E. coli-TOPO strains were cultured. A total of 75 mice were assigned to one of three major groups (n = 25 for each group) and infected with bacteria via oral administration. Two groups were infected with G. hollisae and E. coli-TOPO-tdh to demonstrate their hepatotoxicity; the third group was infected with E. coli-TOPO as a control. For each major group, five subgroups were established (n = 5 for each group) according to treatment dosage . A total of 100 µl of whole blood was withdrawn at 8 different time points: before treatment with bacteria and 4, 8, 16, 32, 64, 128 and 256 hours after bacterial treatment. Blood samples were analyzed for continued liver function . In addition, 6 mice were treated with 1011 organisms/ml of G. hollisae, E. coli-TOPO-tdh, and E. coli-TOPO (n = 2 for each group). For these animals, liver biopsies and H&E staining (200X) were performed 8 hr after bacterial treatment. Finally, 54 mice were treated with G. hollisae, E. coli-TOPO-tdh, and E. coli-TOPO (n = 18 for each group) with a single administration. Within each group, mice were sub-grouped for treatment with bacteria at concentrations of 107, 109, and 1011 organisms/ml (n = 6 for each group). In each concentration group, mice received a PET/CT scan at 8, 72, and 168 hr (n = 2 for each group) after bacterial treatment.An animal infection model was established to evaluate the hepatotoxicity of bacterial infection. The m/z 1024.543 from an SDS-PAGE of Gh-rTDH revealed a unique hit matching 35VSDFWTNR42 of the Gh-rTDH peptide sequence (by MALDI TOF/TOF spectrum)  .−6 µg/ml) caused damage to the liver cells.Liver cell morphology was clearly affected by the administration of Gh-rTDH . In the Gh-rTDH-FITC was bound around the margin of liver cells after the administration of 10 µg/ml Gh-rTDH-FITC for 20 min . MoreoveThe levels of GOT and GPT were not elevated in the control group after the administration of a single dose of PBS. However, the mean GOT and GPT levels were clearly elevated in the group that was treated with 0.1 µg Gh-rTDH, and the highest levels were observed 8 hr after toxin administration. Similar findings were observed in other treatment groups. Higher doses of Gh-rTDH were clearly associated with more severe mouse liver injury .in vivo by the administration of Gh-rTDH.Total bilirubin levels were clearly elevated in the groups that were treated with Gh-rTDH, and an increased dosage of Gh-rTDH resulted in further increases in total bilirubin levels . MoreoveAlbumin levels began to decrease at 32 hr after Gh-rTDH administration, and the decrease was proportional to the dosage. In the groups that were treated with Gh-rTDH, albumin levels progressively decreased and did not recover, even in the 256-hr treatment group . These rCreatinine and CK-MB levels, which reflect kidney and heart injuries, were not elevated in Gh-rTDH-treated mice. The levels of creatinine and CK-MB did not change in proportion to the dosage of Gh-rTDH. The troponin I levels were also normal in all Gh-rTDH-treated mice .No pathological changes were noted in the liver parenchyma of the control group . In mice18F-FDG PET/CT scan. The red color in the merge images indicates 18F-FDG uptake by cells . Higher concentrations of bacteria caused more severe liver injury. Acute hemolytic status, poor albumin synthesis, and more strongly induced immune system function were noted in mice that were fed G. hollisae and E. coli-TOPO-tdh. These patterns are similar to those observed in mice treated with Gh-rTDH. There was no pathological damage to the liver parenchyma in mice treated with E. coli-TOPO  damaged liver cells. We also noted that the MTT assays yielded a similar pattern over 12, 16, 24, and 48 hr under different toxin concentrations. One possible explanation is that when the concentration of toxin increased, cells were not only killed by this toxin but also probably suffered cell division suppression. Therefore, when we prolonged the treatment durations, the number of surviving cells did not clearly differ between the 4 time points. Naim et al. reported that V. parahaemolyticus TDH caused Rat-1 cell injury and that TDH might induce cytotoxicity by acting inside the cells G. hollisae TDH is notably quick and lethal to the liver cells.In this study, both human and mouse liver cells were treated with G. hollisae infection who presented with liver cirrhosis and hepatic encephalopathy in 1982 Vibrio species infections could be more severe in patients with a history of impaired liver function G. hollisae TDH has never been provided. In this study, we found that the GOT and GPT levels in mice treated with Gh-rTDH obviously increased, and acute liver injury was strongly suspected. Muscle injury was not favored as the major reason for the elevation of GOT/GPT, as a 18F-FDG PET/CT scan did not show muscle injury. Moreover, liver biopsy revealed that the periportal areas of the livers were damaged and that the severity of the damage was associated with the dosage of Gh-rTDH. The periportal area in the liver is functionally well-known for its oxidative energy metabolism of fatty acids and amino acids, glucose release and glycogen formation, ammonia detoxification, protective metabolism, and the synthesis of albumin Morros et al. firstly reported an account of a patient suffering a in vitroin vivo arose within 4 hours after treatment with Gh-rTDH and increased in a dose-dependent manner. The acute hemolytic status in vivo results in acute anemia, which would exacerbate tissue hypoxia and organ hypoperfusion. Therefore, septicemia caused by Vibrio species with the tdh gene might be more critical than that caused by the Vibrio species without the tdh gene. Clinically, the hepatotoxicity might be caused via hemolysis. However, the pathological findings revealed that the hepatic injury was mainly located at the periportal areas, and the injury was not diffused. It is suspected that the major etiology is toxin absorption and injury to the liver via the venous return of the portal system.TDH is well-known for having strong hemolytic activity 18F-FDG PET/CT scans have been reported as excellent tools to survey organ metabolism in small animals 18F-FDG PET/CT scans were performed for our assessment. We noted that the uptake of 18F-FDG in the livers decreased in proportion to the administered dosages of Gh-rTDH, which indicate that the hepatic damage in the animals was dose-dependent. In other non-hepatic organs, damage was not obvious.Clinical 18F-FDG gradually increased in trend. We suggest that the livers could finally reconstruct from the destruction of Gh-rTDH exposure, and these liver cells had undergone repair and proliferation via increasing their uptake of glucose, which is well-known as an unavoidable material in metabolism. The metabolism of glucose in the livers damaged by Gh-rTDH almost recovered to a normal range in the 72nd hour after exposure to TDH. Furthermore, the metabolism of glucose crossed the normal range in the 168th hour after exposure to Gh-rTDH, and the recovery was more predominant in mice treated with low dosages than in those treated with a high dosage of Gh-rTDH. The level of glucose uptake crossing the normal range noted that the metabolism of glucose was notably robust in these damaged livers in addition to ongoing strong recovery. According to our findings from the liver biopsies, the construction might be mainly located in the periportal area, which has been labeled as a major location of glucose and amino acid metabolism 18F-FDG intake in the liver during the recovery stage. Overall, this finding might provide strong evidence indicating that the reconstruction of liver continues for at least one week after a single Gh-rTDH exposure and that the damaged liver has the ability to recover from the Gh-rTDH related injury, even when exposed to a massive dosage of Gh-rTDH. Consistent with this observation is the finding that differential hepatotoxicity could be detected when mice were treated with different amounts of G. hollisae and E. coli-TOPO-tdh but were free from hepatotoxicity with E. coli-TOPO. The 18F-FDG PET/CT results of the animal infection models showed that the severity of the liver injury was notably similar in mice treated with 100 µg of Gh-TDH and in mice treated with 1010 organisms of G. hollisae. Therefore, we suspected that 108 organisms of G. hollisae might produce 1 µg of TDH and cause liver injury in vivo. The results clearly demonstrate the in vivo hepatotoxicity of the Gh-tdh gene product.After exposure to Gh-rTDH, the uptake of G. hollisae TDH is reported as having in vitro and in vivo hepatotoxicity in our study. G. hollisae TDH damaged the liver in living animals and mainly attacked the periportal area, which is associated with the synthesis of albumin and the metabolism of glucose. Most importantly, the 18F-FDG PET/CT scan revealed evidence that the reconstruction of the liver continued at least for one week after a single exposure of G. hollisae TDH. Furthermore, the damaged liver was shown to have an adequate ability to recover.In conclusion,


Leptospira spp. present in the blood of patients with leptospirosis during the first week of symptoms can be detected using culture or PCR. A proportion of patients who are positive by PCR are negative by culture. Leptospira spp. are fastidious bacteria, and we hypothesized that a false-negative culture result may represent infection with a distinct bacterial subset that fail to grow in standard culture medium.Pathogenic Leptospira culture and PCR. A single tube nested PCR that amplified a region of the rrs gene was developed and applied, amplicons sequenced and a phylogenetic tree reconstructed.We evaluated our hypothesis during a prospective study of 418 consecutive patients presenting to a hospital in northeast Thailand with an acute febrile illness. Admission blood samples were taken for Leptospira spp., and 81/418 (19%) patients were culture-negative but rrs PCR-positive. The species associated with culture-positive leptospirosis (37 L. interrogans and 2 L. borgpetersenii) were comparable to those associated with culture-negative, PCR-positive leptospirosis .39/418 (9%) patients were culture-positive for Molecular speciation failed to identify a unique bacterial subset in patients with culture-negative, PCR-positive leptospirosis. The rate of false-negative culture was high, and we speculate that antibiotic pre-treatment is the most likely explanation for this. Leptospira .We downloaded and aligned 184 sequence traces from GenBank for the partial or full rrs fragment described above, together with around 50 bases up- and downstream of this target region. PCR primers were designed using PrimerSelect software . Primers were as follows: rrs-outer-F (5' CTCAGAACTAACGCTGGCGGCGCG 3'), rrs-outer-R (5' GGTTCGTTACTGAGGGTTAAAACCCCC3'), rrs-inner-F (5' CTGGCGGCGCGTCTTA 3'), and rrs-inner-R (5' GTTTTCACACCTGACTTACA 3'). The resulting amplicon was 547 bp. A 25 μl PCR reaction contained 4.5 mM of MgCl2, 200 μM of dNTP, 1.25 unit of Tag DNA polymerase , 0.150 pmol of each outer primer, 1.25 pmol of rrs-inner-F, 5 pmol of rrs-inner-R, 1 M of Betaine  and either 1 μl of DNA extracted from laboratory cultures or 5 μl of DNA extracted from EDTA blood samples taken from febrile patients. PCR was performed in duplicate for each sample using a PTC-200 Peltier Thermal Cycler  using the following conditions: one cycle of 95°C for 2 minutes; 40 cycles of 95°C for 10 seconds, 67°C for 15 seconds and 72°C for 30 seconds; 40 cycles of 95°C for 10 seconds, 55°C for 15 seconds and 72°C for 30 seconds; then one cycle of 72°C for 7 minutes. Positive and negative controls were included in each run. The positive control was genomic DNA extracted from L. interrogans serovar Lai strain Lai spiked into human DNA as diluent. Human DNA was extracted from a 5 ml blood sample taken from a single individual and extracted as described above for clinical samples. The negative control was reaction mixture minus DNA template. Amplicons were visualized using 1.5% gel electrophoresis followed by staining with ethidium bromide. A positive PCR result was defined as the visualization of a band of predicted size for one or both samples.Molecular assays were performed at the Mahidol-Oxford Tropical Medicine Research Unit. A single tube nested PCR was developed that would amplify the 443-nucleotide L. interrogans serovar Lai strain Lai and suspended in human DNA as diluent. Genomic equivalents (GE) were estimated based on a genomic size of 4,659,275 bp, the average genome size of two L. interrogans strains [Leptospira spp. .Analytical sensitivity of the assay was determined using 10-fold serial dilutions of genomic DNA extracted from  strains ,14. AnalLeptospira spp. based on a comparison with sequence for 14 pathogenic and intermediate Leptospira spp. acquired from GenBank database using software Mega 5.0 [Purified PCR products were sequenced by Macrogen Inc.  using the rrs-inner-F and rrs-inner-R primers. Sequences were aligned using SeqManII software , and trimmed to the 443-nucleotide region. A neighbor-joining tree was reconstructed prior to inferring Mega 5.0 .rrs fragment of 184 strains revealed a total of 25 unique alleles for 14 different species. Polymorphic sites, a phylogenetic tree and the frequency of each allele by species are shown in Figure Leptospira spp.  each had a single unique rrs type . The remaining 6 species had between two to five alleles; the frequency of each allele within a given species is shown in Figure Alignment of the 443-nucleotide rrs fragment to speciate the 14 Leptospira spp. compared with the nearly whole length rrs gene (median length 1320 bp) for the 184 sequence traces by comparing average (%) nucleotide distances. Average distances ranged from 0.27-2.50% between the pathogenic species and 0.23-3.16% between the intermediate species for the 443-nucleotide region . An LOD value of 1 GE/reaction equates to 40 Leptospira cells per 1 ml of human blood using the DNA extraction and PCR protocol described in materials and methods. Analytical specificity was determined using DNA from 15 Leptospira strains belonging to pathogenic, intermediate or non-pathogenic Leptospira spp. ; the infecting species in these cases  were L. interrogans (n = 37) and L. borgpetersenii (n = 2).Admission blood samples were taken for rrs PCR assay was performed on all 418 samples. This was positive in 37/39 of the culture positive cases. A comparison of the sequence from the two PCR assays demonstrated that the 443-nucleotide rrs fragment amplified directly from blood was identical to the same region of the near-full length rrs gene amplified from cultured organisms in each case. The 443 bp rrs PCR was also positive for 81 of the 379 culture negative cases. The infecting species in these cases were defined based on phylogenetic analysis of the rrs fragment as L. interrogans (n = 76), L. borgpetersenii (n = 4), and an unidentified species   Figure . All 118rrs sequence of the unidentified species from sample L498 clustered in a phylogenetic branch of the tree containing L. borgpertersenii, L. santarosai, L. weilii and L. alexanderi, and was most closely related to L. borgpetersenii   against a panel of reference isolates of the following serovar: Pomona, Hardjo, Tarassovi, Grippotyphosa, Celledoni, Copenhageni, Australis, Pyrogenes, Canicola, Hebdomadis, Mini, Sarmin, Autmnalis, Cynopteri, Ballum, Bataviae, Djasiman, Javanica, Panama, Shermani and Mwalok. The patient became afebrile eight hours after the initiation of doxycycline, made an uneventful recovery, and was discharged home three days after admission. Doxycycline was continued to complete a 7-day course. The patient had made a full clinical recovery when seen in clinic for the day 10 serum sample.Leptospira spp. associated with culture-negative leptospirosis included a distinct subset at the species level compared with Leptospira spp. associated with culture-positive infection. The driver for this was to explain why the majority of patients with PCR-positive leptospirosis in our setting are culture-negative. Demonstration of a specific culture-negative sub-set would have opened the way to develop alternative culture media and potentially increase the sensitivity of culture, but we found no such differences.The purpose of this study was to examine whether Leptospira spp. might also perish in the blood collection tube prior to laboratory culture due to fluctuations in ambient temperature or other factors, and are fastidious bacteria with highly defined growth requirements. Timing of the sample after the onset of symptoms is also important; we have reported previously for the same group of patients that those who were PCR-positive and culture-positive had a shorter duration of illness prior to sampling than those who were PCR-positive but culture-negative [There are several alternative explanations for PCR-positive but culture-negative leptospirosis. The patient may have received an effective antimicrobial drug but may not have cleared non-viable organism by the time that the blood sample was taken. We do not have information on antimicrobial consumption by our study patients prior to hospital admission, and this remains an important possibility. negative . Two patLeptospira spp. and the study population in Thailand may be significantly different to that in other settings.This study has several limitations. The patient group studied were those people who were sufficiently unwell to require hospitalization, and so the findings cannot be generalized to cases with milder clinical manifestations. Furthermore, the characteristics of rrs described here has been noted previously during a study in which a 452-nucleotide region of the 16S ribosomal RNA gene (defined with reference to rrs2 of L. interrogans serovar Copenhageni strain Fiocruz L1-130) was used as one of six loci of a genotyping scheme devised by Ahmed et al. [rrs gene of L. interrogans serovar Lai strain 56601, and includes the 443-nucleotide rrs region used in our study (position 89 to 531). The number of alleles and polymorphic sites in 9 pathogenic and 5 intermediate Leptospira spp. were identical for the 443-nucleotide and 452-nucleotide regions. Ahmed et al. evaluated 120 strains belonging to 6 pathogenic species , which were separated by rrs2 into 29 different alleles containing 20 polymorphic sites. This suggests that alleles of Leptospira rrs arise frequently and could occur through homologous recombination within or between closely related species rather than by mutation [The genetic diversity of d et al. . This remutation .Leptospira species. The genes used previously in such analyses have included 16S rRNA (rrs) [gyrB) [rpoB) [Leptospira species based on genetic differences at one or a small number of genetic loci. At one end of the spectrum, a Leptospira species may be clearly genetically distinct from other members of the genus based on a large degree of genetic diversity. Nucleotide distances between pathogenic and intermediate species evaluated in this study were more that 4% when compared using the nearly entire rrs gene (> 3% based on the 443-nucleotide rrs region). However, defining a species based on genetic differences at a small number of genetic loci is problematic. For example, two closely related Leptospira species (L. interrogans and L. kirschneri) have just 1 to 4 nucleotide differences when compared using the nearly entire gene, and 1 to 3 nucleotide differences based on the 443-nucleotide rrs region. Similarly, the sequence difference between the closely related L. inadai and L. broomii is 3 nucleotides based on the nearly entire gene and a single nucleotide based on the 443-nucleotide rrs region. This is equivalent to the number of differences observed within L. noguchii alone, which using our 443-nucleotide rrs region defined 5 polymorphic sites and 3 alleles. The unidentified species in this study (L498) was most closely related to L. borgpetersenii and had 3 nucleotide differences based on the 443-nucleotide rrs region. It remains to be seen whether this is a new species or a variant of an existing one. The lack of a live bacterial culture in this case precludes further characterization using additional tools such as DNA hybridization.Gene sequence analysis represents an important tool for the delineation of NA (rrs) -29, DNA ) [gyrB) ,30-32 an) [rpoB) ,34. An iLeptospira spp. associated with culture-negative infection, and increases the number of cases for whom the causative species can be identified. This assay also represents a potentially useful epidemiological tool in settings where culture is not available. It is not clear why culture-negative leptospirosis is so common among our patient population, but the ready availability of over the counter antibiotics and the frequency with which these are purchased represents a plausible explanation that could be tested in future studies.The PCR assay described here was designed to speciate The authors declare that they have no competing interests.JT, SP, SB, VW, WC and ND conceived and designed the study. SB and PA performed the laboratory work, SB, JT and DL analyzed the data and JT, SP and SB wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/338/prepubrrs sequences used during primer designAdditional Table Accession numbers of .Click here for file


We show that the expression of lacZ can be imaged by PAT as deep as 5.0 cm in biological tissue, with resolutions of ∼1.0 mm and ∼0.4 mm in the lateral and axial directions, respectively. We further demonstrate non-invasive, simultaneous imaging of a lacZ-expressing tumor and its surrounding microvasculature in vivo by dual-wavelength acoustic-resolution photoacoustic microscopy (AR-PAM), with a lateral resolution of 45 µm and an axial resolution of 15 µm. Finally, using optical-resolution photoacoustic microscopy (OR-PAM), we show intra-cellular localization of lacZ expression, with a lateral resolution of a fraction of a micron. These results suggest that PAT is a complementary tool to conventional optical fluorescence imaging of reporter genes for linking biological studies from the microscopic to the macroscopic scales.Photoacoustic tomography (PAT) is a molecular imaging technology. Unlike conventional reporter gene imaging, which is usually based on fluorescence, photoacoustic reporter gene imaging relies only on optical absorption. This work demonstrates several key merits of PAT using  First, in vitro observation may not correspond to what occurs in vivo due to the difficulty of replicating the native microenvironment. Second, they produce only a single data point from each cell culture or sacrificed animal. To get statistically robust data, procedures must be repeated many times, which is labor-intensive, time-consuming, and costly. Third, they are incapable of localizing where genes are expressed in cells. Fourth, due to the various translational and post-translational modifications of the gene products, the level of mRNA for a gene may not quantitatively correlate with the protein production of the same gene A key finding of the Human Genome Project, completed in 2003, was that in our enormous genome of 20,000–25,000 genes, only 2% of the total encodes all of the proteins necessary for building the human body and executing its physiological functions in vivoWith the promise of overcoming these limitations, molecular imaging is rapidly being developed for interrogating gene expression in vivo due to optical diffusion.Two optical imaging tools, bioluminescence imaging (BLI) and fluorescence imaging (FLI), are currently the main techniques used in preclinical research to study gene expression. BLI can detect fM of protein while FLI has a typical sensitivity on the order of nM in vivo using photoacoustic tomography (PAT) based on optical absorption lacZ-marked 9 L gliosarcoma and its surrounding microvasculature in vivolacZ expression, and also show intra-cellular localization of lacZ expression with a sub-micron lateral resolution. The results indicate that PAT can bridge the gap between the microscopic and macroscopic domains in molecular biology studies.None of the current mainstream molecular imaging modalities can visualize gene expression at both microscopic and macroscopic levels. Previous works have suggested a new approach to imaging gene expression in vivo chromogenic reporter gene systems to solve current challenges in biological imaging.Although developed earlier, chromogenic reporter genes have been much neglected for molecular imaging, largely due to the lack of tools to visualize their expression inside living organisms. PAT remedies this and, in our opinion, will rejuvenate the development of new lacZ gene, one of the most widely used chromogenic reporter genes, for multiscale PAT. Originating from E. Coli, lacZ encodes β-galactosidase, a bacterial enzyme responsible for metabolizing lactose into glucose and galactose. The expression of lacZ can be studied by several chromogenic assays. Among them, we chose to use 5-bromo-4-chloro-3-indolyl-β-D-galactoside  as our in vivo molecular probe. X-gal is a colorless analogue of lactose, in which the glucose fragment is replaced by an indole-derivative. β-galactosidase cleaves the glycosidic linkage in X-gal and yields galactose and an optical transparent indoxyl monomer, 5-bromo-4-chloro-3-hydroxyindole. Afterwards, two of the monomers are oxidized to form a stable insoluble blue product, 5,5′-dibromo-4,4′-dichloro-indigo . Fiin vivo imaging, the lacZ/X-gal reporter gene system has two noticeable advantages. First, X-gal is an activatable probe. X-gal alone is colorless. Strong optical absorption is generated only after it is cleaved by β-galactosidase. Thus, clearance of the un-cleaved X-gal before imaging is not required. As a result, the activity of β-galactosidase can be measured by PAT against a minimal background. Second, this enzymatic reporter system possesses an intrinsic signal-amplification mechanism. As an enzyme, a single β-galactosidase molecule can cleave multiple X-gal molecules to produce a large number of blue product molecules, allowing us to detect a low expression level of lacZ.For lacZ cell line was created by transfecting the 9 L cell with BAG replication deficient retroviral vector carrying the E. Coli lacZ gene. The 9 L/lacZ cells were purchased from American Type Culture Center . The cells were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum  and 1 mM sodium pyruvate (Invitrogen).A wild-type 9 L gliosarcoma cell line was provided by Dr. Michael Welch’s laboratory in the School of Medicine of Washington University in St. Louis . The 9 L/lacZ cells were rinsed with phosphate-buffered saline  and then collected into a 15 ml centrifuge tube. The cells were harvested after centrifuging at 4°C for 5 minutes and discarding the supernatant. Then we mixed ice-cold RIPA lysis buffer  with the cells and incubated them in ice for 40 minutes. During the incubation, the solution was re-suspended every 10 minutes. Finally, the solution was centrifuged for 20 minutes at 4°C. The supernatant, containing the active natively expressed β-galactosidase, was collected as the lysate of 9 L/lacZ cells.After removal of the culture medium, the 9 L/lacZ cells was subcutaneously injected into anaesthetized mice  using a 0.3-ml syringe with a 29-gauge needle. Tumors were left to grow and monitored daily. Imaging was performed after the progression of the tumor became obvious. Hair was removed before imaging. For detecting the expression of lacZ, ∼0.4 mg of X-gal in 20 µl PBS and DMSO (1∶1) was injected near the tumor 1 day before imaging. During imaging, the animals were was maintained under anesthesia by a gas mixture of oxygen and 1% isoflurane flowing at a rate of 1 L/min, and their blood oxygenation and heart rates were closely monitored. All mice recovered naturally after the experiments without photodamage.This animal study was approved by the Washington University in St. Louis Animal Studies Committee. For tumor inoculation, a 5-µl PBS suspension containing 5 million wild-type 9 L or 9 L/2, 10 times lower than the ANSI safety limit (20 mJ/cm2). The acoustic signal was detected by a 128-element linear ultrasonic array with a nominal bandwidth of 4–8 MHz . The imaging probe was scanned along the elevational direction to obtain a volumetric image. For each 2D frame of the photoacoustic image, measurements were averaged for 100 times, requiring an acquisition time of 10 s. Photoacoustic images were reconstructed using a Fourier-domain beam-forming algorithm This system is capable of performing both photoacoustic and ultrasonic imaging of the same sample. The photoacoustic signal was excited by 6.5-ns laser pulses generated at a 10-Hz repetition rate and a wavelength of 650 nm by a tunable dye laser  pumped by a Q-switched Nd:YAG laser . Light fluence on the tissue surface was measured to be ∼2 mJ/cm2. A mechanical stage drove the raster scanning of the imaging probe to obtain a volumetric dataset without averaging. The maximum photoacoustic amplitudes along each axial line were projected onto the skin surface to form a maximum-amplitude projection (MAP) image. The current system was quantified to have a lateral resolution of 45 µm and an axial resolution of 15 µm, and was capable of imaging ∼3 mm deep into the skin A tunable dye laser , pumped by a Nd:YLF laser , provided laser pulses at two wavelengths, 584 nm and 635 nm. The pulse width was 7 ns, and the maximum repetition rate was ∼5 kHz. The light focus coaxially overlapped with the focus of a high-frequency ultrasonic transducer . The incident energy density at the tissue surface was controlled to be below 6 mJ/cm4 laser  generated 1.2-ns pulses at a wavelength of 532 nm. The light was delivered by a single-mode optical fiber and focused into a diffraction-limited spot inside the tissue. The generated photoacoustic signal was detected by an ultrasonic transducer  in transmission mode. The scanning mode was the same as AR-PAM and could also form an MAP image after scanning. The lateral resolution of this set-up was quantified to be 0.40 µm For photoacoustic excitation, a compact diode-pumped Nd:YVOlacZ transgene were grown in its right and left flanks, respectively. Both tumors were injected with the same amount of X-gal before imaging. Once the mouse was sacrificed after imaging, we removed the skin on top of the tumors, and found only the lacZ + tumor was stained blue, which further confirms the specificity of the detection of the lacZ expression by the chromogenic X-gal probe [in vivo with both photoacoustic and ultrasonic imaging. lacZ + tumor in three dimensions. Also, we observed that although the tumor showed as a hypoechoic area in ultrasonic images, the lack of ultrasonic scattering was not a specific indicator for tumor [lacZ/X-gal reporter strategy, PAT could detect the lacZ-marked tumor with a high specificity [lacZ remained visible at a depth of 5.0 cm in biological tissue, with a contrast of 3.04.al probe . The tumor tumor . In contcificity . MoreovelacZ-marked tumor [Blood vessels and the blue product have well-separated absorption peaks, and can be separately visualized by PAT at different optical wavelengths . Two waved tumor , the 584ed tumor . The pholacZ cells grown on a cover glass after staining in 1 mg/ml X-gal solution at 37°C for 12 hours. lacZ cells using a Nanozoomer 2.0-HT slide scanner . We observed that 9 L/lacZ cells were between 20 and 40 µm in diameter. Inside the cells, strong absorbers were scattered around low-absorbing centers that are cell nuclei . This finding implies that β-galactosidase, the final product of lacZ expression, exists mostly in the cytoplasm.lacZ at a depth of 5.0 cm in biological tissue. This demonstrated the advantage of PAT over other optical molecular imaging techniques, such as BLI and FLI, in penetration depth. In addition, all images were obtained by averaging over 100 measurements, but using a low optical energy at 1/10 of the ANSI exposure limit. Assuming noise was uncorrelated between measurements, we could potentially obtain images with the same quality at a speed of 10 frames/second while using the maximum laser energy allowed by the ANSI standard. The imaging speed can be further improved by adopting a high-repetition rate laser.Using the macroscopic photoacoustic system, we imaged the expression of lacZ-marked tumor and its surrounding microvasculature by dual-wavelength AR-PAM. Remodeling of the microcirculation plays an important role in oncology. Although photoacoustic computed tomography is able to detect the lacZ-marked tumor at great depth, it lacks the resolving power to visualize the microcirculation system associated with the tumor. AR-PAM uses a high-frequency focused transducer, and can achieve sufficient resolving power to visualize the microvasculature at the cost of imaging depth.We further demonstrated simultaneous imaging of a It is noted that the reaction product of X-gal is almost insoluble in water and is thus difficult to be cleared by the body. However, by using other galactopyranosides  and ortho-Nitrophenyl-β-galactoside (ONPG)) whose cleavage products by β-galactosidase are water-soluble, chronic monitoring of gene expression in a single sample at multiple time points can be achieved.lacZ expression using OR-PAM. By confining photoacoustic excitation by tight optical focusing, OR-PAM has been proven to obtain a lateral resolution of a fraction of a micron We showed the sub-cellular localization of the Currently, each of the imaging systems is independent, which means one PAT system achieves only one specific resolution. Thus, a single PAT system with switchable resolutions and penetration depths is of great interest. To integrate a macroscopic photoacoustic imaging system with AR-PAM, we simply need to replace the 50-MHz transducer in AR-PAM with a lower frequency transducer and adjust the laser exposure accordingly. Previous work has demonstrated that an AR-PAM with a 5-MHz transducer provides penetration up to 38 mm in chicken breast tissue. At the 19-mm depth, the axial resolution is 144 µm and the lateral resolution is 560 µm lacZ can be detected by PAT as deep as 5.0 cm in biological tissue. In addition, we showed that PAT could follow the gene expression at scalable depths and resolutions. We expect that PAT could become an important tool linking biological studies at the microscopic and macroscopic levels. With the future development of new chromogenic reporter gene systems, photoacoustic reporter gene imaging can impact both laboratory research and clinical practice.In summary, we demonstrated several key merits of PAT as a promising candidate for molecular imaging. We proved that the expression of Video S13D depiction of composite AR-PAM image acquired at 635 nm and 584 nm showing the tumor and blood vessels. Green: tumor. Red: microvasculature.(MPG)Click here for additional data file.


This study examines the downstream NO release pathway and the contribution of different vasodilator mediators in the acetylcholine-induced response in rat aorta 5-months after the loss of ovarian function. Aortic segments from ovariectomized and control female Sprague-Dawley rats were used to measure: the levels of superoxide anion, the superoxide dismutases (SODs) activity, the cGMP formation, the cGMP-dependent protein kinase (PKG) activity and the involvement of NO, cGMP, hydrogen peroxide and hyperpolarizing mechanisms in the ACh-induced relaxation. The results showed that ovariectomy did not alter ACh-induced relaxation; incubation with L-NAME, a NO synthase inhibitor, decreased the ACh-induced response to a lesser extent in aorta from ovariectomized than from control rats, while ODQ, a guanylate cyclase inhibitor, decreased that response to a similar extent; the blockade of hyperpolarizing mechanisms, by precontracting arteries with KCl, decreased the ACh-induced response to a greater extent in aortas from ovariectomized than those from control rats; catalase, that decomposes hydrogen peroxide, decreased the ACh-induced response only in aorta from ovariectomized rats. In addition, ovariectomy increased superoxide anion levels and SODs activity, decreased cGMP formation and increased PKG activity. Despite the increased superoxide anion and decreased cGMP in aorta from ovariectomized rats, ACh-induced relaxation is maintained by the existence of hyperpolarizing mechanisms in which hydrogen peroxide participates. The greater contribution of hydrogen peroxide in ACh-induced relaxation is due to increased SOD activity, in an attempt to compensate for increased superoxide anion formation. Increased PKG activity could represent a redundant mechanism to ensure vasodilator function in the aorta of ovariectomized rats. Vascular endothelium plays a critical role regulating vascular tone by releasing relaxing and contracting factors ; among tIn addition to the activation of the cGMP-PKG pathway, NO may stimulate vascular hyperpolarization . HyperpoClinical studies have shown the important role of endogenous sex hormones in the control of cardiovascular function in men  and womeAlthough modulatory actions of several female sex hormones on NO, cGMP formation -22 and h2 inhalation; the thoracic aorta was carefully dissected out, cleaned of connective tissue, cut into 4 mm long segments and placed in Krebs-Henseleit solution (KHS) at 4 °C containing, in mmol/L: NaCl 115, CaCl2 2.5, KCl 4.6, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 11.1, Na2 EDTA 0.03. The uterus was trimmed and weighed after excess of connective tissue was eliminated, to confirm the effectiveness of ovariectomy. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the USA National Institutes of Health (NIH publication No. 85.23 revised 1985). This study was also approved by the Ethical Committee of the Universidad Autónoma of Madrid.Female Sprague-Dawley rats (6 months old) were divided into two groups: control (in oestrus phase) and ovariectomized rats. All animals were housed in the Animal Facility of the Universidad Autónoma de Madrid (Registration number EX-021U) according to directives 609/86 CEE and R.D. 233/88 of the Ministerio de Agricultura, Pesca y Alimentación of Spain. Bilateral ovariectomy was induced at 4 weeks of age under anesthesia by isoflurane inhalation. The adequacy of anesthesia was tested by observing regular respiratory rhythm and absence of retraction reflex in hind-legs after mechanical stimulation. Rats were treated with 0.30 mg/Kg SC meloxicam  immediately after surgery and with 50 mg/Kg ibuprofen, orally administered for 4 days. Systolic blood pressure was indirectly measured in awake animals by the tail-cuff method  using a in situ, as previously described [Hydroethidine (HE), an oxidative fluorescent dye, was used to evaluate superoxide anion levels escribed . BrieflySuperoxide anion levels were also measured using lucigenin chemiluminescence, as previously described . BrieflyFrozen samples of aortic segments were homogenized in a buffer composed (in mmol/L) of HEPES 20, EGTA 1, mannitol 210 and sucrose 70. After centrifugation at 15000 g for 5 min, 5-10 µl of supernatants were used to the assay. The enzyme activity was measured by a SOD-assay kit  following the manufacturer’s instructions. The SOD activity was expressed as units/ml μg protein.Aortic segments were subjected to a resting tension of 1 g. After an equilibration period of 60 min, segments were contracted with 0.1 µmol/L noradrenaline (NA) during 3 min , and then some segments were incubated with 10 µmol/L ACh for 15 s. Segments were immediately frozen in liquid nitrogen and stored at -70 °C. Levels of cGMP were determined using the cGMP Enzyme Immunoassay Kit from Assay Designs. For this assay, the frozen arteries were homogenized in 0.1 mol/L HCl and centrifuged at 600 g for 10 min at 4 °C. The non-soluble fraction was used to measure protein content with a DC protein assay kit (Bio-Rad). The supernatant was then collected and used for the assay. cGMP levels were measured following the manufacturer’s protocol. Results were expressed as pmol cGMP/mg protein.® Cyclic GMP dependent protein Kinase Assay Kit. The frozen arteries were homogenized in samples prepared according to the manufacturer’s protocol. Non-soluble fractions were used to measure protein content with a DC protein assay kit (Bio-Rad). The supernatant was then collected and used for the assay, following the manufacturer’s instructions. Results were expressed as arbitrary units/mg protein.Aortic segments from control and ovariectomized rats were frozen in liquid nitrogen and stored at -70 °C. PKG activity was measured using the comercial kit CycLex2-5% CO2 mixture (pH 7.4). The segments were subjected to a tension of 1 g which was readjusted every 15 min during a 90 min equilibration period before drug administration. After this, the vessels were exposed to KCl (75 mmol/L) to check their functional integrity. After a washout period, the presence of vascular endothelium was tested by the ability of ACh (10 µmol/L) to relax segments precontracted with NA (0.1 µmol/L).The method used for isometric tension recording has been described in full elsewhere . BrieflyAfter this, the segments were rinsed several times with KHS over 1 h period, and then cumulative concentration-response curves to ACh (0.1 nmol/L -10 µmol/L), to the NO donor sodium nitroprusside  or to the 8Br-cGMP (0.1 µmol/L -10 mmol/L) were obtained in NA-precontracted segments . To analyse the participation of NO or cGMP on the response induced by ACh, the NO synthase inhibitor L-NAME (0.1 mmol/L)  or the gTo study the possible contribution of hyperpolarizing mechanisms to the vasodilator response to ACh or to SNP, some arterial segments were precontracted with 30 mmol/L of KCl before performing the concentration-response curves to ACh or SNP.To analyze the involvement of endogenous hydrogen peroxide in the relaxation induced by ACh or SNP, arteries were incubated with catalase (1000 U/ml) 30 min before the NA- or KCl-induced precontraction and subsequent concentration-response curves to ACh or SNP were performed. μStatistical analysismax value) and the logarithm of the ACh concentration producing 50% of maximum response (log EC50) were calculated by a non-linear regression analysis of each individual concentration-response curve using Graph Pad Prism Software  and the statistical analysis was done used unpaired Student’s t-test. To compare the effect of drugs on ACh-induced responses in aortic segments from control and ovariectomized rats, certain results are expressed as differences in the area under the concentration-response curves (dAUC) between control and experimental conditions. The differences, expressed as a percentage of the control AUC, were analysed using the Student’s t-test. For the experiments on cGMP formation, PKG and SOD activity, the statistical analysis Student’s t-test was also performed. A p value of less than 0.05 was considered significant.Results are given as mean ± S.E.M. (Standard Error of the Mean). The responses elicited by KCl and NA were expressed in mg. The relaxations induced by ACh, SNP or 8Br-cGMP were expressed as a percentage of the initial contraction elicited by NA or KCl. Statistical analysis was done by comparing the curve obtained among groups and in the presence of L-NAME or ODQ with the control curve by means of two-way analysis of variance (ANOVA). The maximum response . Stock solutions (10 mmol/L) of drugs were prepared in distilled water, except for NA which was dissolved in NaCl (0.9%)-ascorbic acid (0.01% w/v) solution. These solutions were kept at -20 °C and appropriate dilutions were made in KHS or HEPES-buffer on the day of the experiment.Cross sections of aorta from control and ovariectomized rat were used to evaluate the presence of superoxide anion. After incubation with hydroethidine, the arteries from ovariectomized rats showed a markedly higher level of EtBr fluorescence than the arteries from control rats. These results indicate that the level of superoxide anion was enhanced by ovariectomy 1.p < 0.001).Similar results were obtained by measuring the chemiluminescence emitted by lucigenin . These results are inversely correlated to those of cGMP formation, indicating the existence of a cGMP-independent mechanism to activate PKG.The activity of PKG was assessed in homogenates from frozen aortas. The assay showed that PKG activity was greater in aortas from ovariectomized than in those of control rats . Likewise, ovariectomy did not modify the contractile response induced by 0.1 µM NA .The exposure of arteries to 75 mmol/L KCl induced a contractile response that was similar in aortas from control and ovariectomized rats , ACh (0.1 nmol/L -10 µmol/L), SNP (0.1 nmol/L -10 µmol/L) or 8Br-cGMP (0.1 µmol/L 0.1 mmol/L) induced a concentration-dependent relaxation that was similar in arteries from control or ovariectomized rats , and similar dAUC after incubation with ODQ in both groups . An additional observation from these results is that L-NAME or ODQ evoked a similar decrease in the ACh-induced response in aortas from control rats, while in aortas from ovariectomized rats ODQ caused a greater inhibition of the ACh-induced relaxation than L-NAME did.To investigate the contribution of NO or cGMP on the vasodilator response induced by ACh (0.1 nmol/L -10 µmol/L), the effect of the inhibitors of NO synthase or guanylate cyclase, L-NAME (0.1 mmol/L) or ODQ (10 µmol/L), respectively, was examined. Preincubation with L-NAME or ODQ significantly decreased the relaxation by ACh in vessels from control and ovariectomized rats compared with the respective untreated arteries 1. In thPrecontraction with KCl (30 mmol/L), that blocks the membrane hyperpolarization, decreased ACh-induced response more in aortas from ovariectomized rats than in those of control rats 1..In NA-precontracted arteries, preincubation with catalase  did not modify the ACh-induced relaxation in aortas from control rats (data not shown), but it decreased the response in arteries from ovariectomized rats 6; in KCIn KCl-precontracted arteries, the relaxation induced by SNP was decreased in similar extent in aortas from both control (data not shown) or ovariectomized rats. In NA-precontracted arteries, the incubation with catalase diminished the SNP-relaxation only in aorta from ovariectomized rats 6, and cIn a previous work, we reported that the loss of ovarian function did not alter the ACh-induced response in rat aorta, despite the overproduction of vasoconstrictor prostanoids which upregulated endothelial NO synthase activity . We alsoin situ detection of superoxide anion. Experiments were undertaken by using hydroethidine fluorescence, as previously reported [in situ is the result of both the production and removal of superoxide anion. Since the elimination of superoxide anion within vessel walls is performed by superoxide dismutases (SODs) that transform superoxide anion to hydrogen peroxide [Vascular function of endothelial NO depends on its bioavailability, which is a balance between NO production and degradation. Reactive oxygen species are involved in metabolizing NO ,26, and reported . We obsereported . This fiperoxide , we analperoxide ,32 and/operoxide  has beenperoxide . This reperoxide , atherosperoxide , orchideperoxide  and/or aperoxide .The elevated levels of superoxide anions metabolize the released endothelial NO. However, we did not observe any modification on NO release, at least in part, because the positive regulation that prostanoids exert on eNOS activity . TherefoAt this point of the discussion, it is interesting to remark the differences obtained between the two groups of animals when the synthesis of NO or cGMP is blocked. Thus, in aorta from control rats both L-NAME and ODQ induced similar degree of blockade of ACh-induced relaxation, which indicates that vascular effect of NO seems to be mediated through cGMP formation. In contrast, in aorta from ovariectomized rats the blockade of cGMP synthesis with ODQ decreased ACh-induced relaxation in greater extent than that induced by inhibiting NO synthesis, which would indicate the existence of vasodilators factors, other than NO, acting in a cGMP-independent way.It is known that the redox environment can modify the activity of different enzymes that participate in the homeostasis of vascular tissues  and thatAs has been discussed earlier, our results also show that aortas from ovariectomized rats produce more superoxide anion than those from control rats, and that SOD activity is also increased in arteries from ovariectomized rats, therefore an increased formation of hydrogen peroxide would be expected. However, enzymes other than SODs cannot be ruled out, since production of hydrogen peroxide from NADPH-oxidase has been reported . Since tIn summary, our data demonstrate that ovariectomy increases the production of superoxide anions and decreases the ACh-induced cGMP formation, which could diminish the ACh-induced relaxation in rat aorta. However, in contrast to that expected, the ACh-induced relaxation is maintained in aortas from ovariectomized rats by the existence of hyperpolarizing mechanisms in which hydrogen peroxide is involved. The greater contribution of hydrogen peroxide to the ACh-induced relaxation is probably due to the increased activity of SOD, in an attempt to compensate for the increased superoxide anion formation. In addition, the activity of PKG is increased in aorta from ovariectomized rats, which could represent a redundant mechanism to ensure vasodilator function in the aorta of these rats see .These results complement those previously reported  and prov


TP53 gene were shown to be associated with inflammation and inflammatory disease. The aim of this study was to investigate the interactions of miR-34b/c and TP53 Arg72-Pro polymorphisms on the risk of IA in a Chinese population. A total of 590 individuals (including 164 patients with IA and 426 controls) were involved in this study. The polymorphisms  were genotyped by polymerase chain reaction-restriction fragment length polymorphism assay and DNA sequencing. We found that the CC genotype of miR-34b/c rs4938723 was significantly associated with a decreased risk of IA compared with the TT genotype. Moreover, a significant gene interaction of the carriers with the combined genotypes of miR-34b/c rs4938723CC and TP53 Arg72Pro CG/CC/GG had a decreased risk of IA, compared with those carrying miR-34b/c rs4938723CT/TT+TP53 Arg72Pro GG/CG/CC combined genotypes. These findings suggest that the miR-34b/c rs4938723CC and TP53 Arg72-Pro polymorphisms may be involved in the susceptibility to IA.Several lines of evidence indicate that inflammatory processes play a key role in the happening and development of intracranial aneurysm (IA). Recently, polymorphisms in the  Intracranial aneurysm (IA) is a common disease with a high prevalence ranging from 1 to 5 percent in large autopsy studies –7. SAH rTP53 gene has an important function in cell cycle control, apoptosis, and maintenance of DNA integrity [TP53, located on chromosome 17p13 [TP53 gene is a frequent functional SNP that leads to a methionine proline conversion [TP53 Arg72Pro SNP results in a change in its protein structure reflected by its altered electrophoretic mobility [TP53 was reported to influence the p53-mediated inflammatory response [Both environmental and genetic factors are involved in the etiology of IA , 23. Sevntegrity –39. The ntegrity . TP53, lme 17p13 , 42 is 1me 17p13 . Codon 7nversion , 45. Themobility , and thiresponse .miR-34 family members, which compose 3 mature miRNAs that are encoded by 2 different pri-miRNAs [miR-34b/c, which may contribute to the susceptibility of HCC in a Chinese population [It is well known that p53 can regulate the expression of miRNAs, especially the i-miRNAs –54. The i-miRNAs . A potenpulation .miR-34b/c and TP53 Arg72-Pro were associated with the risk of IA. To test this hypothesis, we genotyped the 2 SNPs in a case-control study of 164 IA patients and 426 healthy controls in a Chinese population.In this study, we hypothesized that SNPs rs4938723 (T > C) in the promoter region of The study was performed with the approval of the hospital ethics committee, and written informed consent was obtained from all subjects participating in this study. The case-control study population contained 590 unrelated Chinese Han individuals including 164 patients ) with IA and 426 healthy controls ) living in Sichuan province of southwest China. Patients were recruited from the West China Hospital, Sichuan University from January 2008 to September 2009 who were newly diagnosed when they came for emergency because of SAH caused by the rupture of IA or just had general clinical symptoms such as headache or dizziness and diagnosed by DSA . The control group consisted of 426 healthy volunteers from a routine health survey in the same hospital during the same time as the patients. Subjects with any disease in nervous system or other serious illness were intentionally excluded. There was no significant difference between patients and control subjects in age distribution.μL EDTA-anticoagulated peripheral blood using a commercial extraction kit  according to the instruction manual. We used a polymerase chain reaction-restriction fragment length polymorphism assay to detect the genotype of the two SNPs . Primer sequences, reaction conditions, restriction enzymes  used, and length of resulting polymerase chain reaction products have been described previously [Genomic DNA was extracted from 200 eviously . Restrict test. Genotype and allele frequencies of miR-34b/c rs4938723 and TP53 Arg72Pro were obtained using Modified-Powerstates standard edition software. Hardy-Weinberg equilibrium was tested with chi-square test to compare the observed genotype frequencies among the subjects with the expected genotype frequencies. Genotype and allele frequencies of the two SNPs were compared between IA cases and controls using the chi-square tests. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the relative risk conferred by a particular allele, genotype, or the combined genotypes of miR-34b/c rs4938723 and TP53 Arg72Pro. Statistical significance was set at the P < 0.05 level. All the data were analyzed using the SPSS for windows software package version 13.0 .In this retrospective study, demographic and clinical data of both groups were compared by the chi-square test and miR-34b/c rs4938723 and TP53 Arg72Pro were successfully genotyped for 164 patients with IA and 426 controls. The clinical characteristics including sex, age of both groups, and the number of aneurysms in the cases enrolled in our study are shown in miR-34b/c rs4938723 and TP53 Arg72Pro are summarized in Tables miR-34b/c rs4938723 was significantly associated with a decreased risk of IA, compared with the TT genotype . However, no association between TP53 Arg72Pro and the risk of IA was observed for genotypic or allelic association analysis. We also examined the combined effects of miR-34b/c rs4938723 and TP53 Arg72Pro variants on IA risk. As shown in TP53 Arg72Pro CG/CC/GG had a 0.27-fold decreased risk of IA , compared with those carrying rs4938723CT/TT + TP53 Arg72Pro CG/CC/GG combined genotypes, while no other association between combined effects of miR-34b/c rs4938723 and TP53 Arg72Pro variants on IA risk was found. When stratification analysis was done by gender, no association was found (Tables The two SNPs of d Tables .miR-34b/c rs4938723 and TP53 Arg72Pro) and the risk of IA in a Chinese population. We found that the CC genotype of miR-34b/c rs4938723 was significantly associated with a decreased risk of IA, compared with the TT genotype. Moreover, a gene interaction of the carriers with the combined genotypes of miR-34b/c rs4938723CC and TP53 Arg72Pro CG/CC/GG had a decreased risk of IA, compared with those carrying miR-34b/c rs4938723 CT/TT + TP53 Arg72Pro CG/CC/GG combined genotypes. However, no difference was found when stratification analysis was done by gender for both miR-34b/c rs4938723CC and TP53 Arg72Pro.In this study, we investigated the association between two SNPs  synovial tissues [TP53 influences the p53-mediated inflammatory response. The role of p53 in innate immunity and the inflammatory response is now well established [in vivo and the alteration of miR-34b/c under an inflammatory microenvironment can be influenced by p53 [There are two ing site –54. Polying site . Severaling site –62. Whenp53 gene –65. Anot tissues –69. Fran tissues  reportedablished –72 and, ablished . Also a d by p53 .miRNA and p53 gene polymorphisms are associated with IA remain unclear, additional functional studies would provide valuable characterization of the molecular mechanisms by which miRNA and TP53  are involved in susceptibility to IA. However, our study provides evidence that miRNA and TP53 polymorphisms may play an important role in individuals' susceptibility to IA.Although the molecular mechanisms by which There were several limitations in our study. Firstly, detailed lifestyle that may be involved in the occurrence and development of IA was not available. Secondly, the number of subjects in the study was so small which might not be a good representative of the general population. Further studies, therefore, still need to be done.miR-34b/c rs4938723 was significantly associated with a decreased risk of IA, and a significant gene interaction of miR-34b/c rs4938723CT/TT and TP53 Arg72Pro CG/CC/GG was evident on the risk of IA. These findings suggest that miR-34b/c rs4938723 and TP53 Arg72Pro play a role in the formation, process, or rupture of IA. Nevertheless, our findings need to be replicated in larger, preferably multiethic population-based studies.In conclusion, we found that the CC genotype of